Project Summary Chlamydia species are important causes of disease for which no vaccine exists. A fundamental gap in our knowledge is how this obligate intracellular parasite establishes a privileged niche- a membrane bound compartment referred to as the ?inclusion?- in order to survive and replicate within the hostile intracellular environment. Chlamydiae encode a unique class secreted effectors, the Incs (inclusion membrane proteins), inserted directly into the inclusion membrane. Incs are ideally positioned to mediate interactions between the inclusion and the host, and are likely important for Chlamydia's intracellular survival. This grant builds on extensive preliminary studies in which we used large-scale affinity purification/mass spectrometry (AP-MS) to comprehensively identify protein-protein interactions (PPI) between all C. trachomatis Incs and the human proteome. Combined with rigorous bioinformatics analysis, this study identified ~350 high confidence Inc-host PPIs for 38/58 C. trachomatis Incs, representing one of the most comprehensive bacterial-host interactomes to date. Of high interest was the finding that CT192, an early expressed Inc of unknown function, exhibits high confidence interactions with all 11 known subunits of dynactin. This multi-subunit complex regulates the activity of the primary eukaryotic retrograde microtubule (MT)-motor, dynein. Dynactin, together with dynein and cargo adaptor proteins, plays critical roles in many cellular processes, including vesicle and organelle transport along MTs and tethering MTs to the centrosome. Both of these pathways are known to be involved in the C. trachomatis life cycle, and a chemically generated predicted CT192 null mutant exhibits a replication defect in cell culture. Using a combination of proteomic, biochemical, cell biological, and newly developed genetic strategies I will test the hypothesis that interaction between CT192 and dynactin is important for the intracellular life cycle of C. trachomatis. In Aim 1, I will use a combination of proteomics and in vitro processivity assays to map the binding interface between CT192 and dynactin and determine how CT192 regulates dynactin activity, respectively. In Aim 2, I will make use of newly pioneered genetic strategies to create a targeted insertional inactivation of CT192 in C. trachomatis and test the functional role of the CT192- dynactin interaction during infection, as well as determine whether CT192 is a virulence factor in a murine model of genital tract infection. Together these aims will allow me to understand how a Chlamydia effector contributes to the creation of a unique intracellular niche by reprogramming the host. Defining the interaction between CT192 and dynactin at the molecular level will contribute to the understanding of dynactin recruitment and regulation by C. trachomatis, and may provide new insights into dynactin function and regulation broadly applicable to human disease.